DNA strands coding for glycerol-e-phosphate acyltransferase

ABSTRACT

DNA strands having the ability to biotechnologically produce glycerol-3-phosphate acyltransferase (ATase) useful for converting the property of the PG of membrane lipids into that of more chilling resistance, specifically a chimeric gene of glycerol-3-phosphate acyltransferase (ATase) cDNA derived from squash in which the about one-third central region (the site cleaved by Kpn I and Hind III) has been replaced with the corresponding region of spinach ATase cDNA, a cDNA derived from squash in which the about one-sixth central region (the site cleaved by Hind III and Sac I) has been replaced with the corresponding region of spinach ATase cDNA, or a chimeric gene of ATase cDNA derived from spinach in which the about one-third 3&#39;-terminal region (the site cleaved by Kpn I and Eco RI) has been replaced with the corresponding region of squash ATase cDNA are disclosed. 
     These chimeric genes can express a chimeric ATase which has a higher substrate selectivity to unsaturated fatty acids. The DNA strand is introduced and expressed in a chilling sensitive plant, so that it can afford a plant the chilling resistance higher than that afforded by the ATase gene derived from a known chilling resistant plant.

TECHNICAL FIELD

The present invention relates to a DNA strand having the ability tobiotechnologically produce a chimeric glycerol-3-phosphateacyltransferase, referred to hereinafter as ATase, between the two kindsof ATases as produced by a spinach (Spinacea oleracea L.) and a squash(Cucurbita moschata Duch.).

BACKGROUND ART

Lipids constituting the biomembranes of plants changes from the liquidcrystal form into the solid form depending on the lowering ofsurrounding temperature, and the properties of the biomembranes are alsochanged therewith. It is believed in the solid state that the membranelosses the selectivity of material permeability, become incapable ofeffecting the essential functions, and thus the cells are impaired.Among the lipids, phosphatidyl glycerol, referred to hereinafter as PG,is a lipid easily solidified at a high temperature which has a hightransition temperature from the liquid crystal to the solid state. Thus,the sensitivity of the biomembrane to temperature varies depending onthe properties of PG. In this connection, the easy solidificationproperty of PG is determined by the kinds of fatty acids as theconstituents of it. The transfer of the fatty acid toglycerol-3-phosphate, referred to hereinafter as G-3-P, is carried outby G-3-P acyltransferase, referred to hereinafter as ATase, ofchlorophyll. In other words, the transfer reaction of the fatty acidportion from the complex of the fatty acid and an acyl carrier protein,referred to hereinafter as ACP, to the G-3-P is catalyzed by the ATase.

In plants, the synthesis of fatty acids is carried out solely inchlorophyll, and the complex of the fatty acid and the ACP as thesubstrate of the ATase comprises primarily palmitoyl-ACP, referred tohereinafter as 16:0-ACP, and oleoyl-ACP, referred to hereinafter as18:1-ACP. The selection of the substrates by the ATase is determined bythe properties of the ATase itself, that is the substrate selectivity ofthe ATase. The substrate selectivities of the ATase have been examinedin a variety of plants. For example, the ATases of spinach and pea aschilling resistant plants have high substrate specificity to 18:1-ACP,and the PG of these plants are in the liquid crystal state even at arelatively low temperature (Eur. J. Biochem. 129 (1983) 629-636). Bycontrast, the ATase of a chilling sensitive plant such as squash cannotdistinguish 16:0-ACP and 18:1-ACP and transfer the fatty acids inrespective complexes at the substantially equal ratio, so that the PG ofthe squash solidifies at a relatively high temperature (as described indetail below). Further, on measuring the substrate selectivities, theselectivities of fatty acid thioesters can be examined with either caseof using ACP and CoA (Coenzyme A) (Plant Physiol. 83 (1987) 676-680).

Among the ATases of the chilling resistant plants, only the ones ofArabidopsis thaliana (Japanese Patent Laid-Open Publication No.11891/1992; Japanese Patent Application No. 4782/1990), pea (Plant Mol.Biol. 17 (1991) 1067-1076) and spinach disclosed by the present inventor(WO 95/14094, International Application PCT/JP94/01956) have the overallamino acid sequences which have been completely elucidated. It has beenrevealed that the integration of an ATase gene derived from Arabidopsisthaliana as a chilling resistant plant or from squash as a chillingsensitive plant into tobacco as a plant having medium temperaturesensitivity by the technology of genetic engineering permits thetemperature sensitivity of tobacco to change into further chillingresistant in the case of the Arabidopsis thaliana and into furtherchilling sensitive in the case of the squash (Japanese PatentPublication No. 504439/1994; Japanese Patent Application No.502792/1992).

DISCLOSURE OF THE INVENTION

It is known that the substrate selectivities of spinach ATase tounsaturated fatty acid ester (18:1-ACP) are higher than those of pea orArabidopsis thaliana. Also, the amino acid sequences of the ATasederived from the other plants such as squash, cucumber or safflowerwhich are not chilling resistant have been reported. If a gene can becreated which has a substrate selectivity to 18:1-ACP higher than thatof the conventionally known gene for the purpose of affordinglow-temperature resistance to a chilling sensitive plant, it can beexpected that the gene is introduced into the plant to afford strongerchilling resistance to it.

The object of the present invention is to provide a DNA strand havingthe ability to biotechnologically produce an ATase useful for convertingthe PG in membrane lipids into the one having a stronger chillingresistant property.

The inventors have successfully obtained a gene of a chimeric ATaseutilizing an unsaturated fatty acid ester as a substrate and having ahigher reactivity than that of a naturally occurring ATase derived fromspinach by comparing the DNAs and amino acid sequences of spinach as atypical chilling resistant plant and of squash as a chilling sensitiveplant and preparing a gene (chimeric gene) in which these two genes arecombined and blended with each other. The present invention has beenaccomplished on the basis of such informations as described above.

That is to say, the DNA strand having the ability to biotechnologicallyproduce glycerol-3-phosphate acyltranspherase according to the presentinvention is characterized by having a nucleotide sequence encoding apolypeptide with a glycerol-3-phosphate acyltranspherase activity andwith the amino acid sequence corresponding substantially to the aminoacid sequences shown in SEQ ID NOS 1 and 2, 3 and 4, 5 and 6, 7 and 8,or 9 and 10.

Introducing the DNA strand according to the present invention into avariety of plants and expressing the DNA therein make it possible tovary the property of the PG, in a preferred embodiment, to producechilling resistant type of plants such as spinach etc. or the furtherchilling resistant type of plants, that is, to obtain chilling resistantplants. The technique for introducing and expressing the DNA strand inplants is a common technique which has already been conducted in manyplants such as tobacco, petunia, chrysanthemum, carnation, potato andrice.

The present invention also relates to a transformed plant and a processfor preparing it. That is to say, the transformed plant and a processfor preparing it according to the present invention are as follows.

A plant having the content of unsaturated fatty acids in fatty acidsbound to the lipids varied from the original composition owing to theDNA described above incorporated and the glycerol-3-phosphateacyltransferase produced by the expression of the DNA.

A process for varying the composition of the fatty acids in the lipidsin a plant, comprising incorporating the DNA described above into aplant cell and expressing the DNA in the plant to produce theglycerol-3-phosphate acyltransferase, so that the content of unsaturatedfatty acids in fatty acids bound to the lipids in the plant is variedfrom the original composition.

A process for varying the sensitivity of a plant to a low temperature,comprising incorporating the DNA described above into a plant cell andexpressing the DNA in the plant to produce the glycerol-3-phosphateacyltransferase, so that the composition of fatty acids bound to PGcontained in the biomembrane of plant cells is varied, thus varying thecontent of unsaturated molecule species.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is schematic illustrations of ATases of spinach (PPP), squash(QQQ), and primary chimeras.

The chimeric ATases obtained by the DNA strands of the present inventionare QPQ, Q(PQ)Q and PPQ, and the sites recognized by the restrictionenzymes employed for the preparation of chimeras are also illustrated.

FIG. 2 is an illustration, for activities of the naturally occurring andchimeric ATases, shown with raw data (a) and with the relative values(b) of the incorporated amounts of 18:1-CoA into G-3-P to the totalincorporated amounts of 16:0-CoA and 18:1-CoA.

FIG. 3 is the restriction enzyme maps of the chimeric ATase genes QPQ,Q(PQ)Q and PPQ.

Arrows show the directions of translation, and white parts represent theDNA portions derived from spinach with black parts derived from squash.

BEST MODE FOR CARRYING OUT THE INVENTION ATase Genes

Definition

The DNA strand having the ability to biotechnologically produce ATaseaccording to the present invention, that is the ATase gene comprises anucleotide sequence coding for a polypeptide which has the ATaseactivity and amino acid sequence corresponding substantially to the oneof the amino acid sequences shown in SEQ ID NOS 1 and 2, 3 and 4, 5 and6, 7 and 8, or 9 and 10. The term "DNA strand" herein means apolydeoxyribonucleic acid having a certain length. The "DNA strand" inthe invention is specified by the amino acid sequence (including thealtered or modified ones as described hereinafter) of the polypeptidefor which the DNA strand codes, and the polypeptide is limited asdescribed above, so that the "DNA strand" (including the degeneratedisomers as described hereinafter) is limited as well. However, the "DNAstrand" contains the gene coding for the ATase and thus useful for thebiotechnological production of the polypeptide, which is not possiblewith the only DNA strand having the limited length, but possible withthe DNA strand having linked DNA strands having an appropriate lengththereto at the upstream of its 5' side and at the downstream of its 3'side.

Therefore, the term "DNA strand" in the present invention includes inaddition to the DNA strands having the particular lengths (SEQ ID NOS:1, 3, 5, 7 or 9) those in the form of linear or circular DNA strandshaving these DNA strands having the particular lengths as a member.

A typical existence form of the DNA strands according to the presentinvention is a form in which the DNA strand is inserted as a part of themembers in a plasmid or phage DNA, and a form in which the DNA strand ispresent in a microorganism (particularly bacterium), phage particle orplant in the form of being inserted in a plasmid, phage or genomic DNA.It goes without saying that the term bacterium herein includesEscherichia coli and Agrobacterium.

A preferred occurring form of the DNA strands according to the presentinvention is the one present in a plant as a form in which the ATasegene is integrally ligated to components for expression such as apromoter, a DNA strand coding for a translation regulating region, a DNAstrand coding for a transit peptide into chloroplasts, the DNA strandaccording to the present invention, a translation terminating codon anda terminator so that the ATase gene can be stably expressed in theplant, and the integrated DNAs being inserted in a genome. As thecomponents, known components for expression such as a promoter (e.g.cauliflower mosaic virus 35S promoter), a DNA strand coding for atranslation controlling region, a DNA strand coding for a transitpeptide into chloroplasts (e.g. ribulose bisphosphatecarboxylase/oxygenase small subunit), a translation terminating codon, aterminator (e.g. noparin synthase) can be used in an appropriatecombination thereof.

Gene Coding Polypeptide

The DNA strands according to the present invention, as described above,are defined by the amino acid sequences for which the DNA strands code.The polypeptide is a polypeptide which has an ATase activity and whoseamino acid sequence corresponds substantially to the one shown in SEQ IDNOS 1 and 2, 3 and 4, 5 and 6, 7 and 8, or 9 and 10. The phraseology"amino acid sequence corresponds substantially to the one shown in SEQID NOS 1 and 2, 3 and 4, 5 and 6, 7 and 8, or 9 and 10 herein indicatesthat the polypeptide may have a modification or alteration such as adeletion, a substitution, an insertion or an addition for some of theamino acids as long as the polypeptide has the ATase activity.

The ATase as the object in the present invention is an enzyme defined byEC2.3.1.15.

Nucleotide Sequence of the DNA Strand

The DNA strands coding for the ATase according to the present inventionare described above, and their typical examples are the ones having thenucleotide sequences shown in SEQ ID NOS: 1, 3, 5, 7 or 9 or degeneratedisomers thereof as well as the ones having the nucleotide sequencescorresponding to the variation of the amino acid sequence of the ATaseas described above or degenerated isomers thereof. The term "degeneratedisomers" herein means a DNA strand which is different only in thedegenerated codon and can code for the same polypeptide. For example,the DNA strand having the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7or 9 in which a codon corresponding to any one of the amino acids, forexample the codon (AAC) corresponding to Asn has been changed into acodon such as AAT which has a relationship of degeneracy therewith isherein designated a degenerated isomer.

A preferred specific example of the DNA strand according to the presentinvention is a DNA strand having at least one terminating codon (e.g.TAG) flanking the 3'-terminal. Also, a DNA strand in a certain length asa non-translational region may be linked to the upstream of the 5'-sideand/or the downstream of the 3'-side of the DNA strand of the presentinvention.

Acquisition of the DNA Strand

A method for acquiring the DNA strand having the nucleotide sequencewhich codes for the amino acid sequence of the ATase described abovecomprises the chemical synthesis of at least a part of the DNA strandaccording to the method for the synthesis of a nucleic acid.

In consideration of that the number of the amino acid residues of theknown ATase is at least 368, it is preferable to obtain the library of aDNA complementary to mRNA by the conventional method used in the fieldof genetic engineering, for example by the OKAYAMA-BARG method(Molecular Cell Biol. 2 (1982) 161-170) from the mRNA derived from theleaves of spinach and squash rather than by the chemical synthesis, andto create a chimeric gene which is a combination of DNAs derived fromthe both plants after the acquirement with the conventional method suchas the immunological method with an appropriate probe or thehybridization method.

ATase genes have hitherto been isolated from six plants includingspinach and squash, and their DNA structures have been elucidated. Theinventors have cloned the cDNAs of spinach and squash and recombined thetwo genes at the restriction enzyme recognition sites in the DNAsequence common to these two cDNAs to create a chimeric gene between theboth genes.

Specifically, cDNAs derived from spinach and squash were divided intothree approximately equal parts with two restriction enzymes KpnI andHindIII, and chimeric genes of various combinations between both cDNAswere prepared with these divided parts. The inventors have found thatamong a variety of chimeric genes, a chimeric gene in which the middlefragment of the three divided parts of the squash cDNA has been replacedwith the corresponding fragment of the spinach DNA (QPQ, correspondingto SEQ ID NOS: 3 and 4), and a chimeric gene in which the dividedfragment at the 3' terminal of the cDNA of spinach has been replacedwith the corresponding fragment of the cDNA of squash (PPQ,corresponding to SEQ ID NOS: 1 and 2) as well as a chimeric gene inwhich only half at the amino terminal side of the part derived from thecDNA of spinach in the chimeric gene (QPQ) of SEQ ID NOS: 3 and 4 hasremained to be derived from spinach and the remaining half has beenderived from squash (SEQ ID NOS: 5 and 6: Q(PQ)Q) express ATase proteinshaving a high substrate-specificity to unsaturated fatty acids, andaccomplished the present invention on the basis of the informations.

The structure and the method for obtaining the cDNA of the ATase derivedfrom squash are known, and thus the cDNA can be obtained according tothe method (Japanese Patent Laid-Open Publication No. 235594/1989).Also, the structure and the method for obtaining the cDNA of the ATasederived from spinach have been elucidated by the present inventors, andthus the cDNA can be obtained according to the method (WO 95/14094;PCT/JP94/01956).

In addition, the general method for preparing chimeric genes includingthe ligation of fragments of genes can be referred to for exampleMolecular Cloning, Second edition, Sambrook et al. eds., Cold SpringHarbor Laboratory Press, 1989, and specific examples of the preparationof the chimeric genes according to the present invention is illustratedin the examples mentioned hereinafter.

Transformation

As described above, the DNA strands for encoding the chimeric ATasesderived from spinach and squash have been provided according to thepresent invention. In order to express the DNA strands and to producethe polypeptides (ATase) for which the DNA strands code, it is requiredto be introduced into plant cells in the form that in addition to theDNA strands according to the present invention, an appropriate promoter,a DNA strand coding for a transit peptide into chloroplasts, andexpression regulating sequences such as a translation termination codonand a terminator have been ligated integrally. A transformed plant canbe obtained by introducing the DNA strand of the present invention intothe plant cell (transformation) and culturing the cell with anappropriate combination of a promoter such as the 35S promoter of acauliflower mosaic virus, the promoter of a nopaline synthetase, or thepromoter of a small subunit of ribulose-1,5-bisphosphatecarboxylase/oxygenase, and a terminator such as the terminator of thenopaline synthase, or the terminator of an octopine synthase accordingto the known method. It is also possible to employ the known DNA strandfor encoding the transit peptide into chloroplast for example the geneof the small subunit of the ribulose-1,5-bisphosphate carboxylase ofpea.

As described below, the content of unsaturated fatty acids in the fattyacids of PG depending on the differences of the expressions of genes inplant species or strains can be varied (increased or decreased) byintroducing the DNA strands of the present invention into plants, and achilling resistant plant can be obtained by increasing the content ofthe unsaturated fatty acids.

Plants as the object for introducing the DNA strand of the presentinvention may be any one of a chilling sensitive plant, a chillingresistant plant or an intermediate plant of the two, and among theseplants the chilling sensitive plant is particularly preferred.

The chilling resistant plant includes cereals such as rice and corn,potatoes such as taro and sweet potato, vegetables such as cucumber,pimiento, eggplant and squash, fruit trees such as banana and melon,flowering plants such as orpine, cyclamen, lily and rose, and minor farmproducts such as a castor oil plant or sponge gourd. The chillingresistant vegetable and the intermediate vegetable include barley,spinach, pea, Arabidopsis thaliana, tomato and tobacco.

As the method for introducing exogenous genes into plants, variousmethods which have already been reported and established such as themethod in which the Ti plasmid of Agrobacterium is used as a vector, orthe method in which genes are introduced into the protoplast of theplant by electroporation can be used appropriately depending on thevegetables into which genes are intended to be introduced (e.g. see"Plant Molecular Biology Manual" Second edition, S. B. Gelvin and R. A.Schilperoort, Kluwer Academic Publishers, 1995). As the materials ofplants for introducing exogenous genes, it is possible to select theappropriate one from various materials such as a leaf piece, a stempiece, a tuber piece, a protoplast, a callus, a pollen, and a pollentube.

In the preferred embodiment of the present invention, the saturatedmolecular species of phosphatidyl glycerol, i.e. lipid molecular specieswhich cause the phase separation of a biomembrane and thus the chillinginjury to plants can be considerably reduced (the content of unsaturatedmolecular species or unsaturated fatty acids is increased), so that achilling resistant plants are thus obtained.

EXAMPLES

The present invention is now described in more details below withreference to examples, it is not limited to these examples.

Examples Preparation of Chimeric ATase Gene

The cDNAs derived from squash and spinach were prepared according to themethod described above (Japanese Patent Laid-Open Publication No.235594/1989, and WO 95/14094: PCT/JP 94/01956), and cloned at the EcoRIsite of pTZ18R.

(1) Preparation of the ATase Gene of Squash

(i) Acquisition of RNA

The total RNA was obtained by the method described by Chirgwin et al.(Biochemistry 18 (1979) 5294-5299) from about 10 g of cotyledons whichwere obtained by sprouting the seeds of squash in the dark at 30° C. for5 days and irradiating white light for 12 hours. The RNA having poly Awas isolated from the total RNA according to the method described byAviv et al. (Proc. Natl. Acad. Sci. USA, 69 (1972) 1408-1412).

(ii) Preparation of RNA Library Complementary to RNA

The DNA complementary to the above described RNA having poly A wassynthesized according to the method described by Gubler et al. (Gene, 25(1983) 263-269). In this case, oligo (dT) and random oligonucleotideswere used as primers. The double stranded DNA thus synthesized wasmethylated with an EcoRI methylase at the cleavage site by therestriction enzyme EcoRI, and an EcoRI linker (dGGAATTCC; TAKARA SHUZOK.K.) were linked to the ends of the DNA. Furthermore, the extra part ofthe linker was cleaved with a restriction enzyme EcoRI, free linkerswere removed from the cDNA fraction by the gel filtration method, andthe cDNA and a phage λgt11 arm were linked together. The DNA was nextpackaged into λ phage particles by the in vitro packaging method to givea library with λgt11.

(iii) Screening of ATase Gene Retaining Strains

A strain reacting with an antiserum specific to the squash ATase 3 wasselected from the phage library thus obtained as described above toobtain an ATase gene retaining strain.

The cDNA library thus obtained was first infected with the Escherichiacoli, strain Y1090 to search about 150 plates having formed 10,000plaques per plate thereon by the method described by Huynh et al. (DNACloning (1985) IRL, Oxford, Vol. 1, 49-78). Each plate was retained intight contact with a cellulose filter which had been preliminarilydipped with isopropyl β-D-thiogalactopyranoside at a temperature of 37°C. for 2 hours, and then washed three times with 0.15 M NaCl and 50 mMphosphate buffer containing 0.1% Triton X-100 (pH 6.8) for 20 minutes.Next, the antiserum obtained from mice was diluted 1,000 times with thesame buffer as described above, and the nitrocellulose filter wasimpregnated into the dilution and shaken at 4° C. overnight. Thenitrocellulose filter was then washed three times with the bufferdescribed above, reacted with a secondary antibody linked with aperoxidase derived from horseradish thereto at room temperature for 2hours, and washed three times in the same manner as described above.Next, color development was carried out with 4-chloro-1-naphthol andhydrogen peroxide as the substrates, and the transformant strain whichdeveloped a strong color was taken out to carry out secondary selectionwith antibodies. First, the protein produced by each transformant strainwas fixed on a nitrocellulose filter, and it was reacted with anantiserum. The antibodies left on the filter after washing are the oneswhich react only with proteins produced specifically by the transformantstrains. The antibody was dissociated from the filter with 5 mMglycine-HCl (pH 2.3) and 0.15 M NaCl, and subsequently the purifiedATase 3 was subjected to SDS-electrophoresis, followed by the reactionwith the blotted filter. It was judged that the transformant strain inwhich an antibody reacting with the purified ATase 3 has been obtainedis the ATase producing transformant strain. From the strain was prepareda large amount of phage, of which DNA was digested with an restrictionenzyme EcoRI to cut out the foreign DNA, which has a size of about 400bp.

The clone was subjected to nick translation with ³² P-dATP (TAKARA SHUZOK.K.) to prepare a probe having a radioactivity of about 10⁷ dpm/μg. Thecomplementary DNA library was screened again with this probe. The filterhaving adsorbed the phages thereon was remained in a suspensioncontaining 50% formamide, 5× Denhardt's solution (0.1% Ficoll®, 0.1%polyvinylpyrrolidone, 0.1% bovine serum albumin), 5× SSPE (0.75 M NaCl,50 mM sodium phosphate, 5 mM EDTA, pH 7.4), 0.1% SDS and 100 μg/ml ofsalmon sperm DNA overnight at 42° C. The DNA probe labelled with ³² Pwas added for the hybridization for further 24 hours. The filter waswashed according to the conventional method to select a phage whichhybridizes strongly with the probe. It was estimated that this phagecomprises the 1426 bp exogenous DNA in which the 1188 bp open readingframe is present, and that a protein comprising 396 amino acids andhaving a molecular weight of about 44,000 is encoded in this phage. TheEscherichia coli strain (designated AT-03) which has been transformedwith the transformant plasmid pAT-03 obtained by cloning the 1426 bp DNAinto the plasmid vector pTZ18R (Pharmacia) has been deposited intoFermentation Research Institute, Agency of Industrial Science andTechnology, M.I.T.I. (renamed National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology), 1-3,1-chome, Higashi, Tsukuba-Shi, Ibaraki-Ken, Japan, with the acceptancenumber of FERM BP-3094 (deposition date: Mar. 11, 1988)

(2) Preparation of Spinach ATase Gene

(i) Preparation of Spinach cDNA Library

Total RNA was obtained from about 10 g of the cotyledon of spinach(Spinacia oleracea L. var. grabra Viroflay/obtained from Watabe SeedFarm (Miyagi)) according to the method described by Chirgwin et al.[Biochemistry, 18, (1979), 5294-5299]. The RNA possessing poly A [poly(A)⁺ RNA] was isolated from the total RNA according to the methoddescribed by Aviv et al. [Proc. Natl. Acad. Sci. USA, 69, (1972),1408-1412].

DNA (cDNA) complementary to the poly (A)⁺ RNA was synthesized accordingto the method described by Gubler et al. [Gene, 25, (1983), 263-269]. Inthis case, oligo (dT) and random oligonucleotide were used as theprimers. The double stranded cDNA thus synthesized was treated withEcoRI methylase to methylate the cleavage site with the restrictionenzyme EcoRI, and then the EcoRI linker (dGGAATTCC; TAKARA SHUZO K.K.)was added to the both terminals. The surplus of the linker was cut awaywith the restriction enzyme EcoRI, free linkers were removed from thecDNA fraction by gel permeation method, and cDNA was linked to the armof the phage λgt11. Subsequently, the DNA was packaged in the λ phageparticles (Gigapack Gold; Stratagene) to give the spinach cDNA libraryin the phage λgt11.

(ii) Preparation of a Probe for Screening Library

Spinach mRNA was analyzed by the northern blot technique with the cDNAsof the ATases of squash and Arabidopsis thaliana as the probes. The mRNAwas prepared according to the method described above. Five μg of each ofthe poly (A)⁺ RNAs of squash, spinach, barley, rice and pea wasdenaturated with glyoxal, subjected to electrophoresis on 1.5% agarosegel, and the isolated poly (A)⁺ RNA was transferred to a nylon membrane(GeneScreen Plus; DuPont) and hybridized with the cDNA as the probe.Hybridization was carried out in a solution comprising 6× SSPE [1× SSPE:10 mM phosphate buffer (pH 7.0), 1 mM EDTA, 0.15 M NaCl], 0.2% SDS, and100 μg/ml of herring sperm DNA at 60° C. for 16 hours. The membrane wasthen washed with shaking with 2× SSC (1× SSC: 0.15 M NaCl, 15 mM sodiumcitrate) twice at room temperature for 15 minutes and then twice at 42°C. for 15 minutes. As a result, when using the cDNAs of the ATases ofsquash and Arabidopsis thaliana as the probes, an about 2 kb band wasdetected in both of the mRNAs of squash and pea, while no band wasdetected in the mRNA of spinach. It was thus judged difficult to obtainthe cDNA of the ATase of spinach by the screening with use of the cDNAsof the ATases of squash and Arabidopsis thaliana as the probes.

The comparison of the amino acid sequences of the ATases derived fromthese four vegetables (the DNA structures of the ATases derived from theremaining two vegetables have also been elucidated) has revealed thatseveral regions have relatively high homology. Thus, DNA was synthesizedfrom each of such highly homologous region found in the four vegetablesof pea and cucumber in addition to Arabidopsis thaliana and squash, andthe combinations of the two of these DNAs were used as the primers inorder to obtain the DNA inserted by the primers by PCR (Polymerase ChainReaction). The corresponding primer DNAs were synthesized (Model 394DNA/RNA Synthesizer; Applied Biosystems), referred to as primers 1-6,respectively, of which sequences are shown below.

1. (SEQ ID NO:11) 5'-TTGCTGCAGGAATGGAAGAA,

2. (SEQ ID NO:12) 5'-GAGAGCCTTTTGA(T or C)TACTACA,

3. (SEQ ID NO:13) 5'-TGTGTTTATTCGAAAAAGCACATG,

4. (SEQ ID NO:14) 5'-CATGTGCTTTTTTGA(A or G)TAAACACA,

5. (SEQ ID NO:15) 5'-GAAGAAGCATCAAAGGGTGC,

6. (SEQ ID NO:16) 5'-GGAGGGGGCAT(G or T)ATGTCAT.

Among these primers, 1-3 correspond to sense chains, and 4-6 correspondto anti-sense chains. In the PCR reaction, 9 primer sets comprisingcomponents each of which is selected from each group were used. GenomicDNAs or DNAs derived from cDNA library may be used as a template usedfor the PCR reaction. In this case, cDNA was synthesized with a reversetranscriptase and mRNA as a template to form a cDNA/mRNA hybrid, whichwas used as a template. The reaction was carried out with Gen Amp™ RNAPCR Kit (TAKARA SHUZO). The synthesis reaction solution of the firstcDNA strand comprises 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 4 mM DTT, 80mM MgCl₂, 0.8 mM dNTPs, 20 μg/ml of 6mer random oligo DNA, 20 U RNaseinhibitor, 2 μg/ml of poly (A)⁺ RNA, and 50 U of a reverse transcriptaseand amounts to 20 μl in total, on which 100 μl of a mineral oil waslayered for reaction at 42° C. for 1 hour. In a parallel experiment, thesquash mRNA was also used as a template for control in addition to thespinach mRNA. After reaction, the reaction mixture was subjected to atemperature of 95° C. for 5 minutes to inactivate the reversetranscriptase. The aforementioned primers (20 μl) were added to thesynthesis reaction solution of the first cDNA strand in order tosynthesize the second strand. In this case, 35 cycles of PCR werecarried out, with a cycle comprising the reaction at 95° C. for 1minute, at 55° C. for 1 minute and at 72° C. for 2 minutes. Afterreaction, the mineral oil was extracted with 100 μl of chloroform torecover the aqueous layer, which was further treated with 100 μl ofether to remove chloroform. A 10 μl portion of the aqueous layer thusobtained was used for the isolation and analysis of the DNA synthesizedby 1% agarose gel electrophoresis. As a result, an about 300 bpamplified DNA fragment was observed in both cases from spinach andsquash mRNAs only with the primer set of 3 and 6. In the other 8combinations, no band which had the same size as that of the amplifiedDNA fragment derived from the squash mRNA was observed in the DNAderived from the spinach mRNA. The amplified DNA fragment was blunted atthe both terminals with the Klenow fragment and then cloned at the SmaIsite of the plasmid pTZ18R (Pharmacia).

(iii) Screening of ATase cDNA-carrying Strain and Isolation of cDNA

The screening of the cDNA library was carried out with the DNA fragmentthus obtained as a probe. An Escherichia coli strain Y1090 (r-) wasinfected with a transformant phage containing the spinach cDNA toprepare 40 plates having a diameter of about 15 cm on which about 30,000plaques have been formed, and the phage was transcribed to a nylonmembrane (Hybond-N+; Amersham). The probe DNA was labelled with ³²P-dCTP with Multiprime DNA labelling Kit (Amersham). Hybridization wascarried out with a hybridization solution having the same composition asthat described in the paragraph (ii) at 65° C. for 16 hours, and finalwashing was carried out twice with 0.1× SSC at 50° C. for 20 minutes.Positive phages at the first screening was screened again in the samemanner as above to give 3 positive phages, which was then purified inorder to obtain phage DNAs. These phage DNAs were cut with EcoRI, andcDNA was subcloned to the plasmid pTZ18R (Pharmacia) to determine thenucleotide sequence. It was revealed that among the three cDNA clonesthus obtained (SpAT#1-3), SpAT#1 has a length of about 1.5 kbp, and theamino acid sequence of its open reading frame, when compared with theamino acid sequence of the ATases of the other vegetables had an aminoacid deletion at the N-terminal. Thus, the spinach cDNA library wasscreened with SpAT#1 as a probe to give an about 600 bp cDNA clone(SpAT#4). The cDNA clone as the combination of these clones (SpAT#14)has a length of 1,656 bp, in which a 1,413 bp open reading frame ispresent, and it is estimated that a protein comprising 472 amino acidsand having a molecular weight of 52,177 is encoded in the open readingframe.

(3) Preparation of Chimeric ATase Gene

Comparison the nucleotide sequences between the cDNAs derived fromsquash and spinach with a software for analyzing the sequence of DNA andthe like (DNASIS, ver. 3.0) revealed that about 70% of the nucleotidesequence was common on the whole. Several sites of recognizingrestriction enzymes were found in the common sequence, and the sites ofHind III and Kpn I were present at the positions that divide almostequally into three portions (FIG. 1). A part of the ATase gene wassuccessfully obtained by using the combinations of the two of the threeenzymes comprising Eco RI which is the cloning site of the cDNA on thevector and the two restriction enzymes described above. A gene of whichpart was replaced by a gene derived from the other vegetable (chimericgene) was prepared by replacing the part with another gene. In thiscase, genes derived from spinach (referred to as P) and squash (referredto as Q) are now illustrated sequentially from the N-terminal as acombination of the three one-third genes in order to illustrate chimericgenes. According to the illustrating method, natural genes derived fromspinach and squash are illustrated PPP and QQQ, the genes in whichone-third from the N-terminal has been replaced with each other areillustrated as QPP and PQQ, the genes in which the central part has beenreplaced with each other are illustrated PQP and QPQ, and the genes inwhich only the C-terminal has been replaced with each other areillustrated PPQ and QQP (FIG. 1).

In the practical preparation example, the cleavage of the genes derivedfrom spinach and squash with Eco RI produces about 1.7 and 1.4 kbp cDNAfragments, respectively, and the further cutting of these fragments withHind III results in another cleavage at the about one-third positionfrom the N-terminal. The replacement of these parts with each other ledto chimeric genes in which the one-third from the N-terminal had beenreplaced by the gene derived from the other vegetable (QPP and PQQ).

The six chimeric genes thus obtained and the two natural genes (PPP andQQQ) were cloned to the vector pET17b (Novagen) for expression in E.coli, and two DNAs was synthesized in order to remove the transitsequence (Applied Biosystem). In the case of spinach, the following twoDNAs were synthesized:

5'-TGACGCATGCGCTAGCCACTCTCGCACTTATCGTAACGTTCGT-3' (SEQ ID NO:17), or

5'-TGACGCATGCGCTAGCCGTTCTCGCACT-3' (SEQ ID NO:18),

wherein the double underline represents the Sph I site, and the singleunderline represents the Nhe I site; referred to hereinafter assynthetic DNA 1. The former DNA is more preferred.

5'-CAGCTCTTCTGCAGAACGAACGTTACGATA-3' (SEQ ID NO:19),

wherein the swung underline represents the Pst I site. After annealingthe DNAs in the equimolar amount, the fill-in reaction was carried outwith the Klenow fragment of DNA polymerase in the presence of four dNTPsto prepare an adapter having the Nhe I site therein. Also, in the caseof using the latter short synthetic DNA, it was combined with thesynthetic DNA having the Pst I site and used as a primer for PCR inwhich the cDNA of the spinach gene (PPP) was used as a template, and theDNA fragment was amplified with Taq polymerase in the presence of thefour dNTP. In either of these cases, cutting was carried out with Sph Iand Pst I, the DNA fragment was inserted into a plasmid containing theATase cut with the same restriction enzyme set to select a plasmid intowhich the Nhe I site had been introduced. As a result, the amino acidsequence in the neighborhood of the N-terminal of the maturation enzymeof the spinach ATase was changed from Gln-Leu-Leu-Arg (SEQ ID NO:20)into Met-Ala-Ser-His(Arg) (SEQ ID NO:21) (alteration of four aminoacids).

On the other hand, for the gene having the squash ATase at theN-terminal, the site of the restriction enzyme Nhe I was introduced intothe neighborhood of the maturation enzyme of the ATase by PCR. That isto say, after the following two DNAs were synthesized, 30 cycles of PCRwere carried out, with a cycle comprising the reaction at 95° C. for 1minute, at 55° C. for 1 minute and at 72° C. for 2 minutes, to give anabout 100 bp DNA fragment having the Sph I and Nhe I sites at the oneside and the Pst I site at the other side.

5'-ACGGGCATGCGCTAGCCACTCCCGCAAATTTCTCGATGT-3' (SEQ ID NO:22),

wherein the double underline represents the Sph I site, and the singleunderline represents the Nhe I site; referred to hereinafter assynthetic DNA 2, and

5'-CCATTCCTGCAGCAACATTTGGAGGCAGC-3' (SEQ ID NO:23),

wherein the swung underline represents the Pst I site. The DNA fragmentthus obtained was cut with Sph I and Pst I, and inserted into a plasmidcontaining the ATase cut with the same restriction enzyme set to selecta plasmid into which the Nhe I site had been introduced. As a result,the amino acid sequence in the neighborhood of the N-terminal of thematuration enzyme of the squash ATase was changed from Gln-Pro-Ala-His(SEQ ID NO:24) into Met-Ala-Ser-His (SEQ ID NO:21) (alteration of threeamino acids).

Next, there is a Sac I site at the center of the portion Q of thechimera PQP, and a finer chimera for the central part was prepared withthis site. The following DNA was synthesized from the site correspondingto the neighborhood of the center of the spinach gene in the antisensefashion.

5'-ACGAGCTCGGGATCATCATACATGTGCTT-3' (SEQ ID NO:25),

wherein the underline represents the Sac I site; referred to hereinafteras synthetic DNA 3.

Synthetic DNA 1 and 3 were combined, and PCR was carried out under thesame condition as described in the aforementioned example with use ofPPP (natural spinach gene) as a template. The DNA fragment thus producedwas cut with the set of Hind III and Sac I to give a fragment of thecentral part of the gene derived from spinach which ranges from Sac Inewly introduced artificially to Hind III. Also, after PCR with use ofthe squash gene as a template in the same manner as above, the DNAfragment was cut with Hind III and Sac I, and a DNA fragment which hadbeen inserted between the recognition sites of these enzymes wasobtained and recovered. These DNA fragments and a plasmid in which PQPwas cut with the set of restriction enzymes Hind III and Sac I and thefragment between them was removed were combined to prepare P(PQ)P andP(QPQ)P, respectively. In this connection, the sequence derived fromspinach which is present in the center of the latter chimeric gene isthe sequence corresponding to the primer used in PCR. Also, the centralfragment (PQ) of the chimera obtained by cutting P(PQ)P with therestriction enzymes Hind III and Kpn I was inserted in place of P whichhad been removed by digesting QPQ with the same set of enzymes to formQ(PQ)Q.

Plasmids in which the Nhe I site had been introduced at the N-terminalof a variety of chimeric genes thus prepared and natural spinach andsquash genes were cut with Nhe I and Eco RI, and introduced into pET17bdigested with the same set of the restriction enzymes.

Experimental Example 1 Expression of Chimeric ATase Gene in E. coli

The plasmid obtained as described above was introduced into thecompetent cells of the E. coli strain BL21 (DE3)pLysS (Novagen) preparedaccording to the conventional method (Molecular Cloning, pp. 250-251;1981), and a transformant was obtained by the selection according toresistance to ampicillin. E. coli containing only the chimeric gene orthe original plasmid pET17b was cultured in the Luria-Bertani mediumcontaining 100 μg/ml of ampicillin and 30 μg/ml of chloramphenicol at37° C. until absorbance at 600 nm reaches 0.7, andisopropylthiogalactoside (IPTG) was added so as the concentration to be0.4 mM before culturing for further 3 hours. Cells were recovered bycentrifugation and stored at -20° C. until they are used. It has beenconfirmed by measuring the activity according to the method describedbelow that the enzyme activity will not be changed during the storage inthe refrigerator for several days.

The cells having been frozen were thawed on ice, dissolved in a solutioncomprising 20 mM Tris-HCl (pH 8.0), 20 mM dithiothreitol, 10 mM MgCl₂, 1μg/ml of DNase I, and maintained at 4° C. for 1 hour. The E. coli strainBL21 (DE3) pLysS used in the experiment originally produced T7 lysozyme,and thus could be lysed satisfactorily by maintaining it at 4° C. Thelysate was centrifuged at 30,000×g for 10 minutes to isolate thesupernatant, which was further centrifuged at 100,000×g for 1 hour toisolate the supernatant, which was used as the sample for measuring theenzyme activity.

A portion of each sample was subjected to SDS-electrophoresis to examinethe amount of expressed protein. All of the transformants contained theexpressed protein at about 5% of the total sample protein, and theamounts of the protein expressed are not significantly distinguishedbetween chimeric and natural genes.

Experimental Example 2 Measurement of Enzyme Activity

The activity of ATase was measured according to the standard methoddescribed by Bertrams and Heinz [Plant Physiol., 68, (1981), 653-657] bymeasuring the transfer rate from acyl-CoA to [U-¹⁴ C]glycerol-3-phosphate. The standard measurement was carried out at 24° C.with 80 μl of the solution comprising 0.25 M Hepes, pH 7.4, 6 μg/ml ofbovine serum albumin, 0.3 mM [U-¹⁴ C] glycerol-3-phosphate (0.9Ci/mole), about 1 μg of the E. coli extract, and 0.4 mM palmitoyl-CoA oroleyl-CoA. After 8 minutes, 2.3 ml of a mixture of chloroform andmethanol (1:1), 1 ml of 1 M KCl and 0.2 M H₃ PO₄ were added and stirredsufficiently to stop the reaction. After centrifugation, 0.9 ml of thelower layer (organic layer) was recovered and mixed with the cocktail(Aquasol-2) of a liquid scintillation counter to measure the amountincorporated. In this connection, the amount of protein added to thereaction was adjusted so as to be 30% of the amount of proteinexhibiting the maximum amount of incorporation by preliminarilymeasuring the amounts for some concentrations. As a result, while noactivity was detected in the case of E. coli containing pET17b solely,remarkable enzyme activity was detected in the case containing a varietyof chimeric and natural ATase genes (FIG. 2(a)). First, natural PPP andQQQ genes exhibited high activities when an unsaturated fatty acid ester(18:1-CoA) or a saturated fatty acid ester (16:0-CoA), respectively, wasused for the measurement of the activity. In addition, the genes havinga sequence derived from spinach (P) in the central one-third part (PPQ,PPP, QPQ, P(PQ)P, Q(PQ)Q) generally show relatively higher activitieswhen they used unsaturated fatty acid esters, so that this sequence wasthought important for the reactions using unsaturated fatty acids as asubstrate. Furthermore, surprisingly, QPQ, among these genes, showed theactivity 2 times or more as compared with that of the natural gene(PPP), and thus revealed that the enzyme reaction rate of this chimericgene was enhanced by the genetic recombination.

Next, the results of FIG. 2(a) was represented by the relative value tothe both substrates. (FIG. 2(b)). As a result, the chimeric gene PPQused substantially only unsaturated fatty acids as the substrates andthus had a reaction rate to the unsaturated fatty acids faster than thatof the natural gene (PPP). Similarly, chimeric genes QPQ and Q(PQ)Q hada reaction rate to the unsaturated fatty acids faster than that of thenatural gene (PPP). Particularly, from the result of the last chimericgene Q(PQ)Q, the amino-terminal half of the central part was consideredimportant for the reactions using unsaturated fatty acids as asubstrate. Also, any chimera genes have the C-terminal one-third partcomprising a gene derived from squash, and thus a combination of thegene derived from spinach in the central part and the gene derived fromsquash in the C-terminal was thought optimal.

In this connection, FIG. 3 illustrates the restriction enzyme maps ofthe chimeric ATase genes (QPQ and PPQ). In the figure, the arrowillustrates the direction of translation, the clear parts represent DNAparts derived from spinach, and the black parts represent DNA partsderived from squash.

Experimental Example 3 Introduction of a Chimeric ATase Gene (PPQ) intoTobacco Cells

DNA of a chimeric ATase gene (PPQ) was introduced into tobacco cells asdescribed in the following.

(1) Construction of a vector plasmid for expressing in plants

A binary plasmid pBI121 (Clontech) was cut with restriction enzymes SacI and Sma I, and the terminals thus cut were blunted with a Klenowfragment and then ligated with a T4 DNA ligase. The plasmid pBI121(-GUS)thus obtained contains no β-glucuronidase gene (GUS gene) and has thesites of the restriction enzymes Xba I and Bam HI as unique sitesbetween the 35S promoter of a cauliflower mosaic virus and a nopalinesynthase (NOS) terminator.

The plasmid containing the chimeric ATase (PPQ)obtained in Example 1 wascut with a restriction enzyme Eco RI to separate the vector plasmidpTZ18R and the DNA of the chimeric ATase by the low-melting agarose gelelectrophoresis, and the DNA was cut from the gel. Furthermore, theterminal cut of the DNA was blunted with a Klenow fragment. At the sametime, the plasmid pBI121 (-GUS) obtained as described above was cut witha restriction enzyme Bam HI and treated in the same manner as describedabove to give a blunt terminal. The DNA of the chimeric ATase and theplasmid pBI121(-GUS) thus obtained were ligated with a T4 DNA ligase togive a plasmid pBI121-35SPPQ containing a 35S promoter, the DNA of thechimeric ATase and an NOS terminator. In order to replace the 35Spromoter of the plasmid with an NOS promoter, the plasmid was completelycut with Xba I and then decomposed partially with an insufficient amountof Hind III to remove the 35S promoter of about 800 bp. On the otherhand, for the NOS promoter, the following two primers were prepared byPCR with pBI121 as a template. In this connection, for the design of theprimer, the nucleotide sequence of the NOS promoter in pBIN19 wasobtained from the data base (accession number: U09365).

5'-AGAGAAGCTTGATCATGAGCGGAGAATTAA-3' (SEQ ID NO:26),

5'-AGAGTCTAGAGATCCGGTGCAGATTATTTG-3' (SEQ ID NO:27),

wherein the parts of the underline correspond to Hind III and Xbalsites, respectively. The reaction product of about 300 bp was treatedwith these enzymes, and the promoter DNA was purified by low-meltingagarose gel electrophoresis. The DNA fragment and the plasmid havingremoved the 35S promoter therefrom were ligated with a T4 DNA ligase togive a plasmid pBI121-NOSPPQ containing the NOS promoter, the DNA of thechimeric ATase, and the NOS terminator.

(2) Introduction of pBI121-NOSPPQ into Agrobacterium

Agrobacterium tumefaciens LBA 4404 (Clontech) was inoculated into a YEBmedium (5 g/l of beef extract, 2 mM MgSO₄, pH 7.4), cultured at 28° C.for 24 hours, and the culture medium was centrifuged at 3,000 rpm at 4°C. for 20 minutes to collect cells. The cells were washed three timeswith 10 ml of 1 mM HEPES, pH 7.4, once with glycerol, finally suspendedinto 3 ml of 10% glycerol to prepare the agrobacterium cell solution forintroducing DNA.

A 50 μl portion of the Agrobacterium cell solution and the plasmidpBI121-NOSPPQ were placed into a 1 μg cuvette in order to introduce theplasmid DNA into the Agrobacterium by applying electric pulse under thecondition of 25 μF, 2500 V and 200Ω in an electroporation apparatus(Gene Pulser, BioRad). The cell solution was placed into an Eppendorftube, and 800 μl of an SOC medium (20 g/l of tripton, 5 g of yeastextract, 0.5 g of NaCl, 2.5 mM KCl, pH 7.0) was added for static cultureat 28° C. for 1.5 hours. A 50 μl portion of the culture medium wasseeded on a YEB agar medium (1.2% of agar) containing 100 ppm ofkanamycin and cultured at 28° C. for 2 days. A single colony wasselected from the colonies thus obtained, and the plasmid DNA wasprepared from the colony by the alkaline method. After digesting theplasmid DNA with an appropriate restriction enzyme, the DNA fragment wasisolated by 1% agarose gel electrophoresis and confirmed by the Southernblot technique with a ³² P-labelled chimeric ATase DNA as a probe. TheAgrobacterium is referred to as ALBNSPT.

(3) Transformation of Tobacco

The Agrobacterium ALBNSPT thus obtained was shaking cultured in an LBliquid medium containing 50 ppm of kanamycin at 28° C. for 24 hours. A1.5 ml portion of the culture medium was centrifuged at 10,000 rpm for 3minutes to collect cells, washed with 1 ml of the LB medium to removekanamycin, further centrifuged at 10,000 rpm for 3 minutes to collectcells, and suspended again into 1.5 ml of the LB liquid medium to form acell solution for infection.

Next, in order to infect tobacco leaves with Agrobacterium, youngtobacco leaves were collected, dipped into a 0.5% aqueous sodiumhypochlorite solution for 10 minutes, washed three times with sterilewater, and water was wiped off on a sterile filter paper to make leavesfor infection. The leaves were aseptically cut into pieces having a sizeof 1 cm² with a knife, placed on an Agrobacterium cell solution with therear side up, gently shaken for 2 minutes, then placed on a sterilefilter paper to remove the surplus of the Agrobacterium. The suspensionculture cells of tobacco (cultivar: Xanthi-nc) was spread over an MS-B5medium (containing 1.0 ppm of benzyl adenine, 0.1 ppm of naphthaleneacetate and 0.8% agar) (T. Murashige and F. Skoog, Plant Physiol., 15:473, 1962) in a dish, a Whatman No. 1 filter paper (diameter 7.0 cm) waslayered, and the leaves were placed with rear side up on the filterpaper. The dish was sealed with a film sheet and cultured with a lightcycle of lightness for 16 hours and darkness for 8 hours at 25° C. for 2days. Subsequently, the leaves were transferred into an MS-B5 mediumcontaining 250 ppm of CLAPHORAN (Hechst), and cultured in the samemanner for 10 days to remove the agrobacterium. The callus wastransferred to an MS-B5 medium containing 250 ppm of CLAPHORAN and 100ppm of kanamycin, and cultured for further 30 days, during which thecircumference of the leaves was callused, and young plants wereredifferentiated from some of the calluses. The young plant wastransferred onto a (plant hormone free) MS-B5 medium containing 250 ppmof CLAPHORAN and 100 ppm of kanamycin to grow the regenerated cells, andfurther acclimatized to soil for cultivation in a greenhouse. Thetobacco cultivated in the greenhouse was used as a material for thefollowing tests.

Experimental Example 4 Analysis of Fatty Acids in Tobacco into which aGene (PPQ) in the Chimeric ATase has been Introduced

Phosphatidyl glycerols (PG) were prepared from the transformant plantobtained in Example 3 and a control plant (tobacco in which a GUS genehas been introduced by pBI121), and the fatty acids were analyzed.

Extraction of lipids was carried out by the Bligh-Dyer method (Can. J.Biochem. Physiol., 37: 911, 1959). Isopropanol (5 ml) containing 0.1%butylhydroxytoluene were warmed at 80° C., and cells having a wet weightof 2 g were cut into pieces, quickly added to the alcohol, treated at80° C. for 5 minutes and cooled to room temperature. The mixture ofchloroform and methanol (1:2, volume ratio, 20 ml) was added, and afterdisrupting the cells in a homogenizer, the mixture was left standing for15 minutes. The mixture was diluted with 12 ml of chloroform and 12 mlof distilled water, stirred vigorously, separated into the aqueous layerand the organic layer by centrifugation at 3,000 rpm and 4° C. for 30minutes to recover the organic layer (lower layer). An appropriateamount of ethanol was added to the organic layer, and the organicsolvents were removed with a rotary evaporator at 30° C. under reducedpressure. The residue was dissolved in 2 ml of a mixture of chloroformand methanol (1:4, volume ratio) and used as the total lipid extract.

In order to fractionate the lipids, the lipids were mixed with 25 ml ofa suspension of DEAE-Toyopearl 650C (TOSO) and 25 ml of 1 M aqueoussodium acetate solution (pH 7.0) to form an acid type. This was washedsequentially with distilled water and methanol, suspended in methanol,charged into a column having an internal diameter of 2 cm up to a heightof 1.5 cm, and further washed with 50 ml of a mixture of chloroform andmethanol (1:4, volume ratio).

The total lipid extract was applied on the column, washed sequentiallywith 50 ml of a mixture of chloroform and methanol (1:4, volume ratio),50 ml of acetic acid, and 15 ml of a mixture of chloroform and methanol(1:4, volume ratio) to remove most of the contaminated lipids. Then,washing with 50 ml of a 10 M aqueous ammonium acetate solution (20:80,0.2, volume ratio) gave a lipid fraction containing PG. The fraction wasdiluted with 15 ml of ethanol, and the solvents were removed underreduced pressure. The residue was dissolved in 200 μl of a mixture ofchloroform and methanol (2:1, volume ratio), and the lipids wasseparated on a silica gel-TLC plate #5721 (Merck) with a developingsolvent of chloroform:methanol:acetic acid:water (50:20:10:15:5, volumeratio). After TLC separation, primulon was sprayed for fluorescentcoloring under ultraviolet light, and the PG fraction having the samerate of flow as that of the authentic PG was shaven off together withsilica gel and put into a screwed test tube. 2.5 ml of 5% methanolichydrochloric acid was added, and the mixture was reacted to methylatethe fatty acids. The methyl esters of fatty acids were extracted fourtimes with hexane, and the solvent was removed under reduced pressure.Gas chromatography was used for the analysis of fatty acid methylesters. Fractionation was carried out with a gas chromatographGC-17AAWFW (Shimadzu Seisakusho, Ltd.), fatty acid methyl esters wereidentified by comparing the retention times with those of standard fattyacid methyl esters. Quantitative determination was carried out withChromatopak C-R7A plus (Shimadzu Seisakusho, Ltd.). The results areshown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Compositions of fatty acids and molecular species of                          PG in the leaves of tobacco                                                                             Estimated saturated                                 Plant     16:0 + 16:1t + 18:0 (%)                                                                       molecular species (%)                               ______________________________________                                        Control   70.9            41.8                                                Transformant #1                                                                         75.8            51.6                                                Transformant #2                                                                         75.4            50.8                                                Transformant #3                                                                         63.8            27.6                                                Transformant #4                                                                         67.1            34.2                                                Transformant #5                                                                         66.7            33.4                                                ______________________________________                                    

While the content of the saturated fatty acids (16:0+16:1t+18:0) in thefatty acids linked to PG was 70.9% in the control tobacco, the contentincreased in transformants #1 and 2 and decreased in transformants #3, 4and 5 in the tobaccos into which chimeric ATase (PPQ) had beenintroduced. It was thus indicated that the expression of the chimericATase makes possible the both directions of increasing and decreasingthe unsaturated fatty acids. Particularly, in the case of decreasing theunsaturated fatty acid contents, the content of the fatty acids in thetransformant #3 was decreased in a proportion of 7% or more as comparedwith that of the control, and the saturated molecular species was alsodecreased to a level below 28%.

INDUSTRIAL APPLICABILITY

According to the present invention, a chimeric ATase gene having anunsaturated fatty acid ester as a substrate and a higher reactivity thanthat of an ATase derived from naturally occurring spinaches has beensuccessfully obtained by comparing the DNAs and amino acid sequences ofspinach as a typical chilling resistant plant and of squash as achilling sensitive plant and preparing a (chimeric) gene in which thesetwo genes are linked to and blended with each other at the specificregions. (It has been quite an unexpected result that the ATase obtainedfrom the chimeric gene of the combination of a gene derived from achilling resistant plant and a gene derived from a chilling sensitiveplant has an increased substrate selectivity to unsaturated fatty acidsthan that of the chilling resistant plant.

Thus, the DNA strand according to the present invention is useful as agene which is capable of affording a stronger chilling resistance toplants by introducing it into them.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 27                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1104 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1104                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATG GCT AGC CAC TCT CGC ACT TAT CGT AAC GT - #T CGT TCT GCA GAA GAG           48                                                                          Met Ala Ser His Ser Arg Thr Tyr Arg Asn Va - #l Arg Ser Ala Glu Glu           #                 15                                                          - CTG ATA TCT GAA ATA AAA AGG GAA TCA GAA AT - #T GGA AGG TTA CCT AAA           96                                                                          Leu Ile Ser Glu Ile Lys Arg Glu Ser Glu Il - #e Gly Arg Leu Pro Lys           #             30                                                              - AGT GTT GCT TAT GCT ATG GAG GGA CTT TTT CA - #C TAC TAT CGC AAT GCA          144                                                                          Ser Val Ala Tyr Ala Met Glu Gly Leu Phe Hi - #s Tyr Tyr Arg Asn Ala           #         45                                                                  - GTC CTT TCA AGT GGA ATT TCT CAT GCT GAT GA - #A ATA GTG TTG TCA AAC          192                                                                          Val Leu Ser Ser Gly Ile Ser His Ala Asp Gl - #u Ile Val Leu Ser Asn           #     60                                                                      - ATG AGT GTT ATG CTT GAT TTT GTT TTG TTG GA - #T ATT GAG GAC CCT TTT          240                                                                          Met Ser Val Met Leu Asp Phe Val Leu Leu As - #p Ile Glu Asp Pro Phe           # 80                                                                          - GTA TTT CCA CCG TTT CAC AAA GCT ATT CGA GA - #G CCT GCT GAC TAT TAT          288                                                                          Val Phe Pro Pro Phe His Lys Ala Ile Arg Gl - #u Pro Ala Asp Tyr Tyr           #                 95                                                          - TCC TTT GGT CAA GAT TAC ATT CGG CCA TTG GT - #A GAT TTT GGA AAT TCA          336                                                                          Ser Phe Gly Gln Asp Tyr Ile Arg Pro Leu Va - #l Asp Phe Gly Asn Ser           #           110                                                               - TAT GTT GGT AAC ATC GCC ATT TTC CAA GAA AT - #G GAG GAG AAG CTT AAG          384                                                                          Tyr Val Gly Asn Ile Ala Ile Phe Gln Glu Me - #t Glu Glu Lys Leu Lys           #       125                                                                   - CAG GGT GAC AAC ATC ATC TTA ATG TCC AAC CA - #T CAA AGT GAA GCA GAT          432                                                                          Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp           #   140                                                                       - CCC GCA GTG ATT GCA TTA CTT CTG GAG AAG AC - #A AAT TCA CTA ATC GCA          480                                                                          Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GAA AAC TTG ATC TAC ATA GCA GGT GAT CGA GT - #T ATA ACA GAT CCT CTT          528                                                                          Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu           #               175                                                           - TGC AAG CCC TTT AGC ATG GGA AGG AAT CTT CT - #T TGT GTT TAC TCT AAG          576                                                                          Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys           #           190                                                               - AAG CAC ATG TAT GAT GAT CCC GAG CTT GTT GA - #T GTA AAG AAA AGA GCA          624                                                                          Lys His Met Tyr Asp Asp Pro Glu Leu Val As - #p Val Lys Lys Arg Ala           #       205                                                                   - AAT ACA AGG AGT TTG AAA GAG TTG GTC TTA CT - #T TTA AGA GGT GGT TCA          672                                                                          Asn Thr Arg Ser Leu Lys Glu Leu Val Leu Le - #u Leu Arg Gly Gly Ser           #   220                                                                       - AAA ATA ATC TGG ATT GCA CCC AGT GGT GGA AG - #A GAT CGT CCA GAT GCT          720                                                                          Lys Ile Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Ala           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - GTC ACT GGT GAA TGG TAC CCA GCA CCC TTT GA - #T GCT TCT TCA GTG GAC          768                                                                          Val Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp           #               255                                                           - AAC ATG AGA AGG CTT ATT CAA CAT TCG GAT GT - #T CCT GGG CAT TTG TTT          816                                                                          Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe           #           270                                                               - CCC CTT GCT TTA TTA TGT CAT GAC ATC ATG CC - #C CCT CCC TCA CAG GTC          864                                                                          Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val           #       285                                                                   - GAA ATT GAA ATT GGA GAA AAA AGA GTG ATT GC - #C TTT AAT GGG GCG GGT          912                                                                          Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly           #   300                                                                       - TTG TCT GTG GCT CCT GAA ATC AGC TTC GAG GA - #A ATT GCT GCT ACC CAC          960                                                                          Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - AAA AAT CCT GAG GAG GTT AGG GAG GCA TAC TC - #A AAG GCA CTG TTT GAT         1008                                                                          Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp           #               335                                                           - TCT GTG GCC ATG CAA TAC AAT GTG CTC AAA AC - #G GCT ATC TCC GGC AAA         1056                                                                          Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys           #           350                                                               - CAA GGA CTA GGA GCT TCA ACT GCG GAT GTC TC - #T TTG TCA CAA CCT TGG         1104                                                                          Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp           #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 368 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Ala Ser His Ser Arg Thr Tyr Arg Asn Va - #l Arg Ser Ala Glu Glu         #                 15                                                          - Leu Ile Ser Glu Ile Lys Arg Glu Ser Glu Il - #e Gly Arg Leu Pro Lys         #             30                                                              - Ser Val Ala Tyr Ala Met Glu Gly Leu Phe Hi - #s Tyr Tyr Arg Asn Ala         #         45                                                                  - Val Leu Ser Ser Gly Ile Ser His Ala Asp Gl - #u Ile Val Leu Ser Asn         #     60                                                                      - Met Ser Val Met Leu Asp Phe Val Leu Leu As - #p Ile Glu Asp Pro Phe         # 80                                                                          - Val Phe Pro Pro Phe His Lys Ala Ile Arg Gl - #u Pro Ala Asp Tyr Tyr         #                 95                                                          - Ser Phe Gly Gln Asp Tyr Ile Arg Pro Leu Va - #l Asp Phe Gly Asn Ser         #           110                                                               - Tyr Val Gly Asn Ile Ala Ile Phe Gln Glu Me - #t Glu Glu Lys Leu Lys         #       125                                                                   - Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp         #   140                                                                       - Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu         #               175                                                           - Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys         #           190                                                               - Lys His Met Tyr Asp Asp Pro Glu Leu Val As - #p Val Lys Lys Arg Ala         #       205                                                                   - Asn Thr Arg Ser Leu Lys Glu Leu Val Leu Le - #u Leu Arg Gly Gly Ser         #   220                                                                       - Lys Ile Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Val Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp         #               255                                                           - Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe         #           270                                                               - Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val         #       285                                                                   - Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly         #   300                                                                       - Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp         #               335                                                           - Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys         #           350                                                               - Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp         #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1104 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1104                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ATG GCT AGC CAC TCC CGC AAA TTT CTC GAT GT - #T CGC TCT GAA GAA GAG           48                                                                          Met Ala Ser His Ser Arg Lys Phe Leu Asp Va - #l Arg Ser Glu Glu Glu           #                 15                                                          - TTG CTC TCC TGC ATC AAG AAG GAA ACA GAA GC - #T GGA AAG CTG CCT CCA           96                                                                          Leu Leu Ser Cys Ile Lys Lys Glu Thr Glu Al - #a Gly Lys Leu Pro Pro           #             30                                                              - AAT GTT GCT GCA GGA ATG GAA GAA TTG TAT CA - #G AAT TAT AGA AAT GCT          144                                                                          Asn Val Ala Ala Gly Met Glu Glu Leu Tyr Gl - #n Asn Tyr Arg Asn Ala           #         45                                                                  - GTT ATT GAG AGT GGA AAT CCA AAG GCA GAT GA - #A ATT GTT CTG TCT AAC          192                                                                          Val Ile Glu Ser Gly Asn Pro Lys Ala Asp Gl - #u Ile Val Leu Ser Asn           #     60                                                                      - ATG ACT GTT GCA TTA GAT CGC ATA TTG TTG GA - #T GTG GAG GAT CCT TTT          240                                                                          Met Thr Val Ala Leu Asp Arg Ile Leu Leu As - #p Val Glu Asp Pro Phe           # 80                                                                          - GTC TTC TCA TCA CAC CAC AAA GCA ATT CGA GA - #G CCT TTT GAT TAC TAC          288                                                                          Val Phe Ser Ser His His Lys Ala Ile Arg Gl - #u Pro Phe Asp Tyr Tyr           #                 95                                                          - ATT TTT GGC CAG AAC TAT ATA CGG CCA TTG AT - #T GAT TTT GGA AAT TCA          336                                                                          Ile Phe Gly Gln Asn Tyr Ile Arg Pro Leu Il - #e Asp Phe Gly Asn Ser           #           110                                                               - TTC GTT GGT AAC CTT TCT CTT TTC AAG GAT AT - #A GAA GAG AAG CTT AAG          384                                                                          Phe Val Gly Asn Leu Ser Leu Phe Lys Asp Il - #e Glu Glu Lys Leu Lys           #       125                                                                   - CAG GGT GAC AAC ATC ATC TTA ATG TCC AAC CA - #T CAA AGT GAA GCA GAT          432                                                                          Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp           #   140                                                                       - CCC GCA GTG ATT GCA TTA CTT CTG GAG AAG AC - #A AAT TCA CTA ATC GCA          480                                                                          Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GAA AAC TTG ATC TAC ATA GCA GGT GAT CGA GT - #T ATA ACA GAT CCT CTT          528                                                                          Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu           #               175                                                           - TGC AAG CCC TTT AGC ATG GGA AGG AAT CTT CT - #T TGT GTT TAC TCT AAG          576                                                                          Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys           #           190                                                               - AAG CAC ATG TAT GAT GAT CCC GAG CTT GTT GA - #T GTA AAG AAA AGA GCA          624                                                                          Lys His Met Tyr Asp Asp Pro Glu Leu Val As - #p Val Lys Lys Arg Ala           #       205                                                                   - AAT ACA AGG AGT TTG AAA GAG TTG GTC TTA CT - #T TTA AGA GGT GGT TCA          672                                                                          Asn Thr Arg Ser Leu Lys Glu Leu Val Leu Le - #u Leu Arg Gly Gly Ser           #   220                                                                       - AAA ATA ATC TGG ATT GCA CCC AGT GGT GGA AG - #A GAT CGT CCA GAT GCT          720                                                                          Lys Ile Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Ala           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - GTC ACT GGT GAA TGG TAC CCA GCA CCC TTT GA - #T GCT TCT TCA GTG GAC          768                                                                          Val Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp           #               255                                                           - AAC ATG AGA AGG CTT ATT CAA CAT TCG GAT GT - #T CCT GGG CAT TTG TTT          816                                                                          Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe           #           270                                                               - CCC CTT GCT TTA TTA TGT CAT GAC ATC ATG CC - #C CCT CCC TCA CAG GTC          864                                                                          Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val           #       285                                                                   - GAA ATT GAA ATT GGA GAA AAA AGA GTG ATT GC - #C TTT AAT GGG GCG GGT          912                                                                          Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly           #   300                                                                       - TTG TCT GTG GCT CCT GAA ATC AGC TTC GAG GA - #A ATT GCT GCT ACC CAC          960                                                                          Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - AAA AAT CCT GAG GAG GTT AGG GAG GCA TAC TC - #A AAG GCA CTG TTT GAT         1008                                                                          Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp           #               335                                                           - TCT GTG GCC ATG CAA TAC AAT GTG CTC AAA AC - #G GCT ATC TCC GGC AAA         1056                                                                          Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys           #           350                                                               - CAA GGA CTA GGA GCT TCA ACT GCG GAT GTC TC - #T TTG TCA CAA CCT TGG         1104                                                                          Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp           #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 368 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Ala Ser His Ser Arg Lys Phe Leu Asp Va - #l Arg Ser Glu Glu Glu         #                 15                                                          - Leu Leu Ser Cys Ile Lys Lys Glu Thr Glu Al - #a Gly Lys Leu Pro Pro         #             30                                                              - Asn Val Ala Ala Gly Met Glu Glu Leu Tyr Gl - #n Asn Tyr Arg Asn Ala         #         45                                                                  - Val Ile Glu Ser Gly Asn Pro Lys Ala Asp Gl - #u Ile Val Leu Ser Asn         #     60                                                                      - Met Thr Val Ala Leu Asp Arg Ile Leu Leu As - #p Val Glu Asp Pro Phe         # 80                                                                          - Val Phe Ser Ser His His Lys Ala Ile Arg Gl - #u Pro Phe Asp Tyr Tyr         #                 95                                                          - Ile Phe Gly Gln Asn Tyr Ile Arg Pro Leu Il - #e Asp Phe Gly Asn Ser         #           110                                                               - Phe Val Gly Asn Leu Ser Leu Phe Lys Asp Il - #e Glu Glu Lys Leu Lys         #       125                                                                   - Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp         #   140                                                                       - Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu         #               175                                                           - Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys         #           190                                                               - Lys His Met Tyr Asp Asp Pro Glu Leu Val As - #p Val Lys Lys Arg Ala         #       205                                                                   - Asn Thr Arg Ser Leu Lys Glu Leu Val Leu Le - #u Leu Arg Gly Gly Ser         #   220                                                                       - Lys Ile Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Val Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp         #               255                                                           - Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe         #           270                                                               - Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val         #       285                                                                   - Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly         #   300                                                                       - Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp         #               335                                                           - Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys         #           350                                                               - Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp         #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1104 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1104                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - ATG GCT AGC CAC TCC CGC AAA TTT CTC GAT GT - #T CGC TCT GAA GAA GAG           48                                                                          Met Ala Ser His Ser Arg Lys Phe Leu Asp Va - #l Arg Ser Glu Glu Glu           #                 15                                                          - TTG CTC TCC TGC ATC AAG AAG GAA ACA GAA GC - #T GGA AAG CTG CCT CCA           96                                                                          Leu Leu Ser Cys Ile Lys Lys Glu Thr Glu Al - #a Gly Lys Leu Pro Pro           #             30                                                              - AAT GTT GCT GCA GGA ATG GAA GAA TTG TAT CA - #G AAT TAT AGA AAT GCT          144                                                                          Asn Val Ala Ala Gly Met Glu Glu Leu Tyr Gl - #n Asn Tyr Arg Asn Ala           #         45                                                                  - GTT ATT GAG AGT GGA AAT CCA AAG GCA GAT GA - #A ATT GTT CTG TCT AAC          192                                                                          Val Ile Glu Ser Gly Asn Pro Lys Ala Asp Gl - #u Ile Val Leu Ser Asn           #     60                                                                      - ATG ACT GTT GCA TTA GAT CGC ATA TTG TTG GA - #T GTG GAG GAT CCT TTT          240                                                                          Met Thr Val Ala Leu Asp Arg Ile Leu Leu As - #p Val Glu Asp Pro Phe           # 80                                                                          - GTC TTC TCA TCA CAC CAC AAA GCA ATT CGA GA - #G CCT TTT GAT TAC TAC          288                                                                          Val Phe Ser Ser His His Lys Ala Ile Arg Gl - #u Pro Phe Asp Tyr Tyr           #                 95                                                          - ATT TTT GGC CAG AAC TAT ATA CGG CCA TTG AT - #T GAT TTT GGA AAT TCA          336                                                                          Ile Phe Gly Gln Asn Tyr Ile Arg Pro Leu Il - #e Asp Phe Gly Asn Ser           #           110                                                               - TTC GTT GGT AAC CTT TCT CTT TTC AAG GAT AT - #A GAA GAG AAG CTT AAG          384                                                                          Phe Val Gly Asn Leu Ser Leu Phe Lys Asp Il - #e Glu Glu Lys Leu Lys           #       125                                                                   - CAG GGT GAC AAC ATC ATC TTA ATG TCC AAC CA - #T CAA AGT GAA GCA GAT          432                                                                          Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp           #   140                                                                       - CCC GCA GTG ATT GCA TTA CTT CTG GAG AAG AC - #A AAT TCA CTA ATC GCA          480                                                                          Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GAA AAC TTG ATC TAC ATA GCA GGT GAT CGA GT - #T ATA ACA GAT CCT CTT          528                                                                          Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu           #               175                                                           - TGC AAG CCC TTT AGC ATG GGA AGG AAT CTT CT - #T TGT GTT TAC TCT AAG          576                                                                          Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys           #           190                                                               - AAG CAC ATG TAT GAT GAT CCC GAG CTC ACA GA - #A ACA AAA AGG AAA GCA          624                                                                          Lys His Met Tyr Asp Asp Pro Glu Leu Thr Gl - #u Thr Lys Arg Lys Ala           #       205                                                                   - AAC ACA CGA AGT CTT AAG GAG ATG GCT TTA CT - #C TTA AGA GGT GGA TCA          672                                                                          Asn Thr Arg Ser Leu Lys Glu Met Ala Leu Le - #u Leu Arg Gly Gly Ser           #   220                                                                       - CAA CTA ATA TGG ATT GCA CCC AGT GGT GGT AG - #G GAC CGG CCG GAT CCC          720                                                                          Gln Leu Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Pro           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - TCG ACT GGA GAA TGG TAC CCA GCA CCC TTT GA - #T GCT TCT TCA GTG GAC          768                                                                          Ser Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp           #               255                                                           - AAC ATG AGA AGG CTT ATT CAA CAT TCG GAT GT - #T CCT GGG CAT TTG TTT          816                                                                          Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe           #           270                                                               - CCC CTT GCT TTA TTA TGT CAT GAC ATC ATG CC - #C CCT CCC TCA CAG GTC          864                                                                          Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val           #       285                                                                   - GAA ATT GAA ATT GGA GAA AAA AGA GTG ATT GC - #C TTT AAT GGG GCG GGT          912                                                                          Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly           #   300                                                                       - TTG TCT GTG GCT CCT GAA ATC AGC TTC GAG GA - #A ATT GCT GCT ACC CAC          960                                                                          Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - AAA AAT CCT GAG GAG GTT AGG GAG GCA TAC TC - #A AAG GCA CTG TTT GAT         1008                                                                          Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp           #               335                                                           - TCT GTG GCC ATG CAA TAC AAT GTG CTC AAA AC - #G GCT ATC TCC GGC AAA         1056                                                                          Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys           #           350                                                               - CAA GGA CTA GGA GCT TCA ACT GCG GAT GTC TC - #T TTG TCA CAA CCT TGG         1104                                                                          Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp           #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 368 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Met Ala Ser His Ser Arg Lys Phe Leu Asp Va - #l Arg Ser Glu Glu Glu         #                 15                                                          - Leu Leu Ser Cys Ile Lys Lys Glu Thr Glu Al - #a Gly Lys Leu Pro Pro         #             30                                                              - Asn Val Ala Ala Gly Met Glu Glu Leu Tyr Gl - #n Asn Tyr Arg Asn Ala         #         45                                                                  - Val Ile Glu Ser Gly Asn Pro Lys Ala Asp Gl - #u Ile Val Leu Ser Asn         #     60                                                                      - Met Thr Val Ala Leu Asp Arg Ile Leu Leu As - #p Val Glu Asp Pro Phe         # 80                                                                          - Val Phe Ser Ser His His Lys Ala Ile Arg Gl - #u Pro Phe Asp Tyr Tyr         #                 95                                                          - Ile Phe Gly Gln Asn Tyr Ile Arg Pro Leu Il - #e Asp Phe Gly Asn Ser         #           110                                                               - Phe Val Gly Asn Leu Ser Leu Phe Lys Asp Il - #e Glu Glu Lys Leu Lys         #       125                                                                   - Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp         #   140                                                                       - Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu         #               175                                                           - Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys         #           190                                                               - Lys His Met Tyr Asp Asp Pro Glu Leu Thr Gl - #u Thr Lys Arg Lys Ala         #       205                                                                   - Asn Thr Arg Ser Leu Lys Glu Met Ala Leu Le - #u Leu Arg Gly Gly Ser         #   220                                                                       - Gln Leu Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Pro         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ser Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp         #               255                                                           - Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe         #           270                                                               - Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val         #       285                                                                   - Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly         #   300                                                                       - Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp         #               335                                                           - Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys         #           350                                                               - Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp         #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1104 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1104                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - CAA CTT CTT CGT TCT CGC ACT TAT CGT AAC GT - #T CGT TCT GCA GAA GAG           48                                                                          Gln Leu Leu Arg Ser Arg Thr Tyr Arg Asn Va - #l Arg Ser Ala Glu Glu           #                 15                                                          - CTG ATA TCT GAA ATA AAA AGG GAA TCA GAA AT - #T GGA AGG TTA CCT AAA           96                                                                          Leu Ile Ser Glu Ile Lys Arg Glu Ser Glu Il - #e Gly Arg Leu Pro Lys           #             30                                                              - AGT GTT GCT TAT GCT ATG GAG GGA CTT TTT CA - #C TAC TAT CGC AAT GCA          144                                                                          Ser Val Ala Tyr Ala Met Glu Gly Leu Phe Hi - #s Tyr Tyr Arg Asn Ala           #         45                                                                  - GTC CTT TCA AGT GGA ATT TCT CAT GCT GAT GA - #A ATA GTG TTG TCA AAC          192                                                                          Val Leu Ser Ser Gly Ile Ser His Ala Asp Gl - #u Ile Val Leu Ser Asn           #     60                                                                      - ATG AGT GTT ATG CTT GAT TTT GTT TTG TTG GA - #T ATT GAG GAC CCT TTT          240                                                                          Met Ser Val Met Leu Asp Phe Val Leu Leu As - #p Ile Glu Asp Pro Phe           # 80                                                                          - GTA TTT CCA CCG TTT CAC AAA GCT ATT CGA GA - #G CCT GCT GAC TAT TAT          288                                                                          Val Phe Pro Pro Phe His Lys Ala Ile Arg Gl - #u Pro Ala Asp Tyr Tyr           #                 95                                                          - TCC TTT GGT CAA GAT TAC ATT CGG CCA TTG GT - #A GAT TTT GGA AAT TCA          336                                                                          Ser Phe Gly Gln Asp Tyr Ile Arg Pro Leu Va - #l Asp Phe Gly Asn Ser           #           110                                                               - TAT GTT GGT AAC ATC GCC ATT TTC CAA GAA AT - #G GAG GAG AAG CTT AAG          384                                                                          Tyr Val Gly Asn Ile Ala Ile Phe Gln Glu Me - #t Glu Glu Lys Leu Lys           #       125                                                                   - CAG GGT GAC AAC ATC ATC TTA ATG TCC AAC CA - #T CAA AGT GAA GCA GAT          432                                                                          Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp           #   140                                                                       - CCC GCA GTG ATT GCA TTA CTT CTG GAG AAG AC - #A AAT TCA CTA ATC GCA          480                                                                          Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GAA AAC TTG ATC TAC ATA GCA GGT GAT CGA GT - #T ATA ACA GAT CCT CTT          528                                                                          Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu           #               175                                                           - TGC AAG CCC TTT AGC ATG GGA AGG AAT CTT CT - #T TGT GTT TAC TCT AAG          576                                                                          Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys           #           190                                                               - AAG CAC ATG TAT GAT GAT CCC GAG CTT GTT GA - #T GTA AAG AAA AGA GCA          624                                                                          Lys His Met Tyr Asp Asp Pro Glu Leu Val As - #p Val Lys Lys Arg Ala           #       205                                                                   - AAT ACA AGG AGT TTG AAA GAG TTG GTC TTA CT - #T TTA AGA GGT GGT TCA          672                                                                          Asn Thr Arg Ser Leu Lys Glu Leu Val Leu Le - #u Leu Arg Gly Gly Ser           #   220                                                                       - AAA ATA ATC TGG ATT GCA CCC AGT GGT GGA AG - #A GAT CGT CCA GAT GCT          720                                                                          Lys Ile Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Ala           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - GTC ACT GGT GAA TGG TAC CCA GCA CCC TTT GA - #T GCT TCT TCA GTG GAC          768                                                                          Val Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp           #               255                                                           - AAC ATG AGA AGG CTT ATT CAA CAT TCG GAT GT - #T CCT GGG CAT TTG TTT          816                                                                          Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe           #           270                                                               - CCC CTT GCT TTA TTA TGT CAT GAC ATC ATG CC - #C CCT CCC TCA CAG GTC          864                                                                          Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val           #       285                                                                   - GAA ATT GAA ATT GGA GAA AAA AGA GTG ATT GC - #C TTT AAT GGG GCG GGT          912                                                                          Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly           #   300                                                                       - TTG TCT GTG GCT CCT GAA ATC AGC TTC GAG GA - #A ATT GCT GCT ACC CAC          960                                                                          Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - AAA AAT CCT GAG GAG GTT AGG GAG GCA TAC TC - #A AAG GCA CTG TTT GAT         1008                                                                          Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp           #               335                                                           - TCT GTG GCC ATG CAA TAC AAT GTG CTC AAA AC - #G GCT ATC TCC GGC AAA         1056                                                                          Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys           #           350                                                               - CAA GGA CTA GGA GCT TCA ACT GCG GAT GTC TC - #T TTG TCA CAA CCT TGG         1104                                                                          Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp           #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 368 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Gln Leu Leu Arg Ser Arg Thr Tyr Arg Asn Va - #l Arg Ser Ala Glu Glu         #                 15                                                          - Leu Ile Ser Glu Ile Lys Arg Glu Ser Glu Il - #e Gly Arg Leu Pro Lys         #             30                                                              - Ser Val Ala Tyr Ala Met Glu Gly Leu Phe Hi - #s Tyr Tyr Arg Asn Ala         #         45                                                                  - Val Leu Ser Ser Gly Ile Ser His Ala Asp Gl - #u Ile Val Leu Ser Asn         #     60                                                                      - Met Ser Val Met Leu Asp Phe Val Leu Leu As - #p Ile Glu Asp Pro Phe         # 80                                                                          - Val Phe Pro Pro Phe His Lys Ala Ile Arg Gl - #u Pro Ala Asp Tyr Tyr         #                 95                                                          - Ser Phe Gly Gln Asp Tyr Ile Arg Pro Leu Va - #l Asp Phe Gly Asn Ser         #           110                                                               - Tyr Val Gly Asn Ile Ala Ile Phe Gln Glu Me - #t Glu Glu Lys Leu Lys         #       125                                                                   - Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp         #   140                                                                       - Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu         #               175                                                           - Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys         #           190                                                               - Lys His Met Tyr Asp Asp Pro Glu Leu Val As - #p Val Lys Lys Arg Ala         #       205                                                                   - Asn Thr Arg Ser Leu Lys Glu Leu Val Leu Le - #u Leu Arg Gly Gly Ser         #   220                                                                       - Lys Ile Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Val Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp         #               255                                                           - Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe         #           270                                                               - Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val         #       285                                                                   - Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly         #   300                                                                       - Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp         #               335                                                           - Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys         #           350                                                               - Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp         #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1104 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1104                                               -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - GAG CCG GCT CAC TCC CGC AAA TTT CTC GAT GT - #T CGC TCT GAA GAA GAG           48                                                                          Glu Pro Ala His Ser Arg Lys Phe Leu Asp Va - #l Arg Ser Glu Glu Glu           #                 15                                                          - TTG CTC TCC TGC ATC AAG AAG GAA ACA GAA GC - #T GGA AAG CTG CCT CCA           96                                                                          Leu Leu Ser Cys Ile Lys Lys Glu Thr Glu Al - #a Gly Lys Leu Pro Pro           #             30                                                              - AAT GTT GCT GCA GGA ATG GAA GAA TTG TAT CA - #G AAT TAT AGA AAT GCT          144                                                                          Asn Val Ala Ala Gly Met Glu Glu Leu Tyr Gl - #n Asn Tyr Arg Asn Ala           #         45                                                                  - GTT ATT GAG AGT GGA AAT CCA AAG GCA GAT GA - #A ATT GTT CTG TCT AAC          192                                                                          Val Ile Glu Ser Gly Asn Pro Lys Ala Asp Gl - #u Ile Val Leu Ser Asn           #     60                                                                      - ATG ACT GTT GCA TTA GAT CGC ATA TTG TTG GA - #T GTG GAG GAT CCT TTT          240                                                                          Met Thr Val Ala Leu Asp Arg Ile Leu Leu As - #p Val Glu Asp Pro Phe           # 80                                                                          - GTC TTC TCA TCA CAC CAC AAA GCA ATT CGA GA - #G CCT TTT GAT TAC TAC          288                                                                          Val Phe Ser Ser His His Lys Ala Ile Arg Gl - #u Pro Phe Asp Tyr Tyr           #                 95                                                          - ATT TTT GGC CAG AAC TAT ATA CGG CCA TTG AT - #T GAT TTT GGA AAT TCA          336                                                                          Ile Phe Gly Gln Asn Tyr Ile Arg Pro Leu Il - #e Asp Phe Gly Asn Ser           #           110                                                               - TTC GTT GGT AAC CTT TCT CTT TTC AAG GAT AT - #A GAA GAG AAG CTT AAG          384                                                                          Phe Val Gly Asn Leu Ser Leu Phe Lys Asp Il - #e Glu Glu Lys Leu Lys           #       125                                                                   - CAG GGT GAC AAC ATC ATC TTA ATG TCC AAC CA - #T CAA AGT GAA GCA GAT          432                                                                          Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp           #   140                                                                       - CCC GCA GTG ATT GCA TTA CTT CTG GAG AAG AC - #A AAT TCA CTA ATC GCA          480                                                                          Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - GAA AAC TTG ATC TAC ATA GCA GGT GAT CGA GT - #T ATA ACA GAT CCT CTT          528                                                                          Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu           #               175                                                           - TGC AAG CCC TTT AGC ATG GGA AGG AAT CTT CT - #T TGT GTT TAC TCT AAG          576                                                                          Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys           #           190                                                               - AAG CAC ATG TAT GAT GAT CCC GAG CTT GTT GA - #T GTA AAG AAA AGA GCA          624                                                                          Lys His Met Tyr Asp Asp Pro Glu Leu Val As - #p Val Lys Lys Arg Ala           #       205                                                                   - AAT ACA AGG AGT TTG AAA GAG TTG GTC TTA CT - #T TTA AGA GGT GGT TCA          672                                                                          Asn Thr Arg Ser Leu Lys Glu Leu Val Leu Le - #u Leu Arg Gly Gly Ser           #   220                                                                       - AAA ATA ATC TGG ATT GCA CCC AGT GGT GGA AG - #A GAT CGT CCA GAT GCT          720                                                                          Lys Ile Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Ala           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - GTC ACT GGT GAA TGG TAC CCA GCA CCC TTT GA - #T GCT TCT TCA GTG GAC          768                                                                          Val Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp           #               255                                                           - AAC ATG AGA AGG CTT ATT CAA CAT TCG GAT GT - #T CCT GGG CAT TTG TTT          816                                                                          Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe           #           270                                                               - CCC CTT GCT TTA TTA TGT CAT GAC ATC ATG CC - #C CCT CCC TCA CAG GTC          864                                                                          Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val           #       285                                                                   - GAA ATT GAA ATT GGA GAA AAA AGA GTG ATT GC - #C TTT AAT GGG GCG GGT          912                                                                          Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly           #   300                                                                       - TTG TCT GTG GCT CCT GAA ATC AGC TTC GAG GA - #A ATT GCT GCT ACC CAC          960                                                                          Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - AAA AAT CCT GAG GAG GTT AGG GAG GCA TAC TC - #A AAG GCA CTG TTT GAT         1008                                                                          Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp           #               335                                                           - TCT GTG GCC ATG CAA TAC AAT GTG CTC AAA AC - #G GCT ATC TCC GGC AAA         1056                                                                          Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys           #           350                                                               - CAA GGA CTA GGA GCT TCA ACT GCG GAT GTC TC - #T TTG TCA CAA CCT TGG         1104                                                                          Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp           #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 368 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Glu Pro Ala His Ser Arg Lys Phe Leu Asp Va - #l Arg Ser Glu Glu Glu         #                 15                                                          - Leu Leu Ser Cys Ile Lys Lys Glu Thr Glu Al - #a Gly Lys Leu Pro Pro         #             30                                                              - Asn Val Ala Ala Gly Met Glu Glu Leu Tyr Gl - #n Asn Tyr Arg Asn Ala         #         45                                                                  - Val Ile Glu Ser Gly Asn Pro Lys Ala Asp Gl - #u Ile Val Leu Ser Asn         #     60                                                                      - Met Thr Val Ala Leu Asp Arg Ile Leu Leu As - #p Val Glu Asp Pro Phe         # 80                                                                          - Val Phe Ser Ser His His Lys Ala Ile Arg Gl - #u Pro Phe Asp Tyr Tyr         #                 95                                                          - Ile Phe Gly Gln Asn Tyr Ile Arg Pro Leu Il - #e Asp Phe Gly Asn Ser         #           110                                                               - Phe Val Gly Asn Leu Ser Leu Phe Lys Asp Il - #e Glu Glu Lys Leu Lys         #       125                                                                   - Gln Gly Asp Asn Ile Ile Leu Met Ser Asn Hi - #s Gln Ser Glu Ala Asp         #   140                                                                       - Pro Ala Val Ile Ala Leu Leu Leu Glu Lys Th - #r Asn Ser Leu Ile Ala         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Glu Asn Leu Ile Tyr Ile Ala Gly Asp Arg Va - #l Ile Thr Asp Pro Leu         #               175                                                           - Cys Lys Pro Phe Ser Met Gly Arg Asn Leu Le - #u Cys Val Tyr Ser Lys         #           190                                                               - Lys His Met Tyr Asp Asp Pro Glu Leu Val As - #p Val Lys Lys Arg Ala         #       205                                                                   - Asn Thr Arg Ser Leu Lys Glu Leu Val Leu Le - #u Leu Arg Gly Gly Ser         #   220                                                                       - Lys Ile Ile Trp Ile Ala Pro Ser Gly Gly Ar - #g Asp Arg Pro Asp Ala         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Val Thr Gly Glu Trp Tyr Pro Ala Pro Phe As - #p Ala Ser Ser Val Asp         #               255                                                           - Asn Met Arg Arg Leu Ile Gln His Ser Asp Va - #l Pro Gly His Leu Phe         #           270                                                               - Pro Leu Ala Leu Leu Cys His Asp Ile Met Pr - #o Pro Pro Ser Gln Val         #       285                                                                   - Glu Ile Glu Ile Gly Glu Lys Arg Val Ile Al - #a Phe Asn Gly Ala Gly         #   300                                                                       - Leu Ser Val Ala Pro Glu Ile Ser Phe Glu Gl - #u Ile Ala Ala Thr His         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Lys Asn Pro Glu Glu Val Arg Glu Ala Tyr Se - #r Lys Ala Leu Phe Asp         #               335                                                           - Ser Val Ala Met Gln Tyr Asn Val Leu Lys Th - #r Ala Ile Ser Gly Lys         #           350                                                               - Gln Gly Leu Gly Ala Ser Thr Ala Asp Val Se - #r Leu Ser Gln Pro Trp         #       365                                                                   - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                # 20               AGAA                                                       - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #21                CTAC A                                                     - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #                24AGCA CATG                                                  - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #                24TAAA CACA                                                  - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                # 20               GTGC                                                       - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                # 19               CAT                                                        - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                # 43               CACT CTCGCACTTA TCGTAACGTT CGT                             - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #             28   CGTT CTCGCACT                                              - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #           30     CGAA CGTTACGATA                                            - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Gln Leu Leu Arg                                                             - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Met Ala Ser His                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #    39            CACT CCCGCAAATT TCTCGATGT                                  - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                #            29    ATTT GGAGGCAGC                                             - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 4 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Gln Pro Ala His                                                             1                                                                             - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                #            29    CATA CATGTGCTT                                             - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #           30     GAGC GGAGAATTAA                                            - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: other nucleic acid                                  #= "Primer"A) DESCRIPTION: /desc                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                #           30     GTGC AGATTATTTG                                            __________________________________________________________________________

What is claimed is:
 1. A chimeric DNA that codes for an enzyme havingglycerol-3-phosphate acyltransferase activity, the enzyme comprising afirst partial amino acid sequence selected from the group consisting ofposition 126 (Lys) to 201 (Leu) of SEQ ID NO: 1, position 126 (Lys) to201 (Leu) of SEQ ID NO: 2, position 126 (Lys) to 201 (Leu) of SEQ ID NO:3, position 126 (Lys) to 201 (Leu) of SEQ ID NO: 4, position 126 (Lys)to 201 (Leu) of SEQ ID NO: 5, position 126 (Lys) to 201 (Leu) of SEQ IDNO: 6, position 126 (Lys) to 201 (Leu) of SEQ ID NO: 7, position 126(Lys) to 201 (Leu) of SEQ ID NO: 8, position 126 (Lys) to 201 (Leu) ofSEQ ID NO: 9, and position 126 (Lys) to 201 (Leu) of SEQ ID NO: 10, anda second partial amino acid sequence selected from the group consistingof position 245 (Trp) to 368 (Trp) of SEQ ID NO: 1, position 345 (Trp)to 368 (Trp) of SEQ ID NO: 2, position 245 (Trp) to 368 (Trp) of SEQ IDNO: 3, position 245 (Trp) to 368 (Trp) of SEQ ID NO: 4, position 245(Trp) to 368 (Trp) of SEQ ID NO: 5, position 245(Trp) to 368 (Trp) ofSEQ ID NO: 6, position 245 (Trp) to 368 (Trp) of SEQ ID NO: 7, position245(Trp) to 368 (Trp) of SEQ ID NO: 8, position 245 (Trp) to 368 (Trp)of SEQ ID NO: 9, and position 245 (Trp) to 368 (Trp) of SEQ ID NO: 10,wherein the enzyme has a higher substrate selectivity foroleoyl-(acyl-carrier-protein) than for palmitoyl-(acyl-carrier-protein).2. A chimeric DNA as described in claim 1, wherein the DNA sequenceencodes a polypeptide having an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQID NO:
 10. 3. A chimeric DNA according to claim 1, wherein thenucleotide sequence encoding the polypeptide is selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 andSEQ ID NO: 5, or a degenerated isomer thereof.
 4. A plant having analtered unsaturated fatty acid composition, wherein the plant comprisesa chimeric DNA strand according to claim 1 and the DNA expressesglycerol-3-phosphate acyltransferase activity that leads to the alteredunsaturated fatty acid composition.
 5. A plant according to claim 4,wherein the content of the unsaturated fatty acids is increased withcomparison to a plant that lacks the chimeric DNA.
 6. A process forvarying the composition of fatty acids in plant lipids, comprisingincorporating DNA as described in claim 1 into a plant cell andexpressing the DNA in the plant to produce glycerol-3-phosphateacyltransferase activity.
 7. A process for varying the sensitivity of aplant to a low temperature, comprising incorporating DNA according toclaim 1 into a plant cell and expressing the DNA to produceglycerol-3-phosphate acyltransferase, wherein the glycerol-3-phosphateacyltransferase varies the composition of fatty acids bound tophosphatidyl glycerol compared to fatty acids within a plant that lacksthe incorporated DNA.
 8. A process according to claim 6, wherein thecontent of the unsaturated fatty acids is increased.
 9. A processaccording to claim 7, wherein the content of the unsaturated fatty acidsis increased.